Tetrakis(p‐tolyl)oxalamidinato‐bis[acetylacetonatopalladium(II)] ([Pd2(acac)2(oxam)]) reacted with Li–C≡C–C6H5 in THF with formation of [Pd(C≡C–C6H5)4Li2(thf)4] ( 1a ). Reaction of [Pd2(acac)2(oxam)] with a mixture of 6 equiv. Li–C≡C–C6H5 and 2 equiv. LiCH3 resulted in the formation of [Pd(CH3)(C≡C–C6H5)3Li2(thf)4] ( 2 ), and the dimeric complex [Pd2(CH3)4(C≡C–C6H5)4Li4(thf)6] ( 3 ) was isolated upon reaction of [Pd2(acac)2(oxam)] with a mixture of 4 equiv. Li–C≡C–C6H5 and 4 equiv. LiCH3. 1 – 3 are extremely reactive compounds, which were isolated as white needles in good yields (60–90%). They were fully characterized by IR, 1H‐, 13C‐, 7Li‐NMR spectroscopy, and by X‐ray crystallography of single crystals. In these compounds Li ions are bonded to the two carbon atoms of the alkinyl ligand. 1a reacted with Pd(PPh3)4 in the presence of oxygen to form the already known complexes trans‐[Pd(C≡C–C6H5)2(PPh3)2] and [Pd(η2‐O2)(PPh3)2]. In addition, 1a is an active catalyst for the Heck coupling reaction, but less active in the catalytic Sonogashira reaction. 相似文献
The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila.
Results
In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1ΔPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1ΔPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.
Conclusion
Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable. 相似文献
The central part of the cover picture shows that the rates for long‐range charge transfer across covalent phenylene bridges can be very sensitive to the chemical substituents attached to the individual bridging units. The peripheral parts of the picture illustrate the flash–quench technique employed to investigate intramolecular charge transfer between a phenothiazine donor and a photochemically generated ruthenium(III) complex. On page 1203 , M. E. Walther and O. S. Wenger explain their experimental findings in terms of donor‐bridge energy matching for hole transfer.
We present a technique for measuring colloid size distributions between 15 nm and 2 microm at concentrations relevant to natural surface waters. Two particle-measuring methods are combined: laser-induced breakdown detection (LIBD), which allows the quantification of colloid size distributions below 400 nm, and a commercial single-particle counter that extends the accessible size range up to two mum. Centrifugation was used in order to separate micrometer sized particles for the LIBD measurement. The feasibility is demonstrated on water of Lake Brienz (Switzerland) and the River Pfinz (Germany) and the particle size distributions follow Pareto's law even down to 15 nm in both cases. 相似文献
Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3'-end of primers were corrected and detected within the corresponding amplicons. 相似文献
Nucleocapsids of herpesviruses originate in the nucleus of host cells and bud through the inner nuclear membrane acquiring tegument and envelope. The release of the enveloped virus particle from the perinuclear space is unknown. Cryobased electron microscopic imaging revealed enveloped virus particles within cisterns associated with the perinuclear space, a pre-Golgi compartment connecting Golgi cisterns to the perinuclear space, and enveloped virus particles in Golgi cisterns where they are packaged into transport vacuoles by membrane fission. To our knowledge, our images show for the first time the connectivity from the perinuclear space to Golgi cisterns. The data strongly indicate an intracisternal transport of enveloped virus particles from the budding site to the packaging site. Budding starts by condensation at the inner membrane. Condensation involving the viral envelope and peripheral tegument was persistent in virus particles within perinuclear space and associated cisterns. Virus particles within Golgi cisterns and transport vacuoles originating by Golgi membrane fission, however, lacked condensation. Instead, spikes were clearly evident. The phenomenon of condensation is considered likely to be responsible for preventing fusion of the viral envelope with cisternal membranes and/or for driving virions from the perinuclear space to Golgi cisterns. Glycoprotein K is discussed to likely play a role in the intracisternal transportation of virions. In addition to the pathway including intracisternal transport and packaging, there were clear indications for the well-known pathway involving wrapping of cytoplasmic nucleocapsids by Golgi membranes. The origin of the cytoplasmic nucleocapsids, however, remains obscure. Lack of evidence for release of nucleocapsids at the outer nuclear membrane suggests that the process is very rapid, or that nucleocapsids pass the nucleocytoplasmic barrier via an alternative route. 相似文献
An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product. 相似文献
BACKGROUND: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. RESULTS: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the omega end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. CONCLUSIONS: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms. 相似文献